Optical systems and methods for biological analysis

ABSTRACT

An instrument for processing and/or measuring a biological process contains a sample processing system, an excitation source, an excitation optical system, an optical sensor, and an emission optical system. The sample processing system is configured to retain a first sample holder and a second sample holder, wherein the number of sample cells is different for each sample holder or a characteristic dimension for the first sample cells is different from that of the second sample holder. The instrument also includes an excitation source temperature controller comprising a temperature sensor that is coupled to the excitation source. The temperature controller is configured to produce a first target temperature when the first sample holder is retained by the instrument and to produce a second target temperature when the second sample holder is retained by the instrument.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 14/766,725, filed Aug. 7, 2015, which is a U.S. 371 national stageof International Application No. PCT/US2014/018110, filed Feb. 24, 2014,and claims priority to U.S. Application No. 61/768,367, filed Feb. 22,2013, each of which are incorporated herein by reference in theirentirety.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates generally to systems, devices, and methodsfor observing, testing, and/or analyzing one or more biological samples,and more specifically to systems, devices, and methods comprising anoptical system for observing, testing, and/or analyzing one or morebiological samples.

2. Description of the Related Art

Optical systems for biological and biochemical reactions have been usedto monitor, measure, and/or analyze such reactions in real time. Suchsystems are commonly used in sequencing, genotyping, polymerase chainreaction (PCR), and other biochemical reactions to monitor the progressand provide quantitative data. For example, an optical excitation beammay be used in real-time PCR (qPCR) reactions to illuminatehybridization probes or molecular beacons to provide fluorescent signalsindicative of the amount of a target gene or other nucleotide sequence.Increasing demands to provide greater numbers of reactions per test orexperiment have resulted in instruments that are able to conduct everhigher numbers of reactions simultaneously.

The increase in the number sample sites in a test or experiment has ledto microtiter plates and other sample formats that provide ever smallersample volumes. In addition, techniques such as digital PCR (dPCR) haveincreased the demand for smaller sample volumes that contain either zeroor one target nucleotide sequence in all or the majority of a largenumber of test samples. The combination of small feature size (e.g., anindividual sample site or volume) and large field of view to accommodatea large number of test samples has created a need for optical systemsthat provide high optical performance with relatively small samplesignals.

The reduction in sample volumes has also lead to a desire to incorporatelight sources that provide a large amount output power or intensity. Inrecent years, advance in LED (Light Emitting Diode) technology resultedin availability of LED sources with significantly larger outputs. Inaddition, high power LED sources are now available with a broadspectrum, for example, white light LEDs that provide significant outputpower across the visible spectrum. Broad spectrum or white light LEDsare also attractive in biological applications such as PCR, since theyallow for a broad range of dyes or markers to be used in a single sampleor instrument. However, high power LEDs can have large power andspectral variations from the nominal specification. Thus, various LEDshaving the same part number or output specification may result inunacceptably large instrument to instrument variation, particularlywould couple with other system tolerance variation (e.g., variations infilter and beamsplitter optical characteristics). Thus, there exists aneed for better control and calibration systems, devices, and methodswhen attempting to incorporate high power, broad spectrum LED intobiological instruments.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention may be better understood from thefollowing detailed description when read in conjunction with theaccompanying drawings. Such embodiments, which are for illustrativepurposes only, depict novel and non-obvious aspects of the invention.The drawings include the following figures:

FIG. 1 is a schematic representation of a system according to anembodiment of the present invention.

FIG. 2 is a table of the filter functions for a plurality of filter usedin the emission filter assembly shown in FIG. 1

FIG. 3 is a table of the filter functions for a plurality of filter usedin the excitation filter assembly shown in FIG. 1

FIG. 4 is a top view of a sample holder and carrier according to anembodiment of the present invention.

FIG. 5 is a top view and magnified views of a sample holder according toanother embodiment of the present invention.

FIG. 6 is a perspective view of a sample holder according to yet anotherembodiment of the present invention.

FIG. 7 is a top view of a sample holder and carrier according to anotherembodiment of the present invention.

FIG. 8 is a graph showing the spectral output from an excitation sourceaccording to an embodiment of the present invention.

FIG. 9 is a cross-sectional view of a heated lid, sample holder, andcarrier according on an embodiment of the present invention.

FIG. 10 is a cross-sectional view of a heated lid, sample holder, andcarrier according on another embodiment of the present invention.

FIG. 11 is a block diagram of a computer system according to anembodiment of the present invention.

FIG. 12 is a plot showing reference temperature verses different ambientand fan conditions for an LED.

FIG. 13 is a plot showing reference temperature verses different ambientand fan conditions for an LED.

FIG. 14 is a plot showing reference temperature verses different ambientand fan conditions for an LED.

FIG. 15 is a flow chart of a method according to an embodiment of thepresent invention.

FIG. 16 is a flow chart of a method according to an embodiment of thepresent invention.

DETAILED DESCRIPTION OF THE DRAWINGS

As used herein, the term “light” means electromagnetic radiation withinthe visible waveband, for example, electromagnetic radiation with awavelength in a vacuum that is within a range from 390 nanometers to 780nanometers. As used herein, the term “infrared” means electromagneticradiation having a wavelength within a range of 0.74 micrometer to 300micrometers.

As used herein, the term “optical power” means the ability of a lens oroptic to converge or diverge light to provide a focus (real or virtual)when disposed within air. As used herein the term “focal length” meansthe reciprocal of the optical power. As used herein, the term“diffractive power” or “diffractive optical power” means the power of alens or optic, or portion thereof, attributable to diffraction ofincident light into one or more diffraction orders. Except where notedotherwise, the optical power of a lens, optic, or optical element isfrom a reference plane associated with the lens or optic (e.g., aprincipal plane of an optic).

As used here, the term “about zero” or “approximately zero” means within0.1 of the unit of measure being referred to, unless otherwise noted.For example, “about zero meters” means less than or equal to 0.1 meters,if the dimension may only reasonably have a positive value, or within arange of −0.1 meters to +0.1 meters, if the dimension may have either apositive or negative value.

When used in reference to an optical power in units of Diopters, theterms “about” or “approximately”, as used herein, means within 0.1Diopter. As used herein, the phrase “about zero Diopter” or“approximately zero Diopter” means within a range of −0.1 Diopter to+0.1 Diopters.

Referring to FIGS. 1-3, a system or instrument 1000 for biologicalanalysis comprises an optical system 100. In certain embodiments, systemor instrument 1000 additionally comprises a sample block or processingsystem 200 and/or a computer system, electronic processor, controller400 configured to control, monitor, and/or receive data from opticalsystem 100 and/or sample processing system 200. Without limiting thescope of the present invention, system or instrument 1000 may be asequencing instrument, a polymerase chain reaction (PCR) instrument(e.g., a real-time PCR (qPCR) instrument and/or digital PCR (dPCR)instrument), an instrument for providing genotyping information, or thelike.

In certain embodiments, optical system 100 comprises an illumination orexcitation source 110 providing one or more excitation beams 111 and anoptical sensor or detector 118 configured to receive one or moreemission beams 119 from one or more biological samples 115. Excitationsource 110 may comprise, or operate in conjunction with, an excitationsource temperature controller 112, which may be used to maintain thetemperature of excitation source 110 above or below a predeterminedtemperature and/or within a predetermined temperature range. Opticalsystem 100 also comprises an excitation optical system 120 and anemission optical system 125. Excitation optical system 120 is disposedalong an excitation optical path 126 and is configured to direct theelectromagnetic radiation or light from excitation source 110 to sampleholder containing one or more biological samples. Emission opticalsystem 125 is disposed along an emission optical path 128 and isconfigured to direct electromagnetic emissions from biological samples115 to optical sensor 118, for example, one or more fluorescence signalsproduced at one or more wavelengths in response to the one or moreexcitation beams 111. Optical system 100 may further comprise anemission filter assembly 130 comprising a plurality of filters, filtercomponents, elements, or modules 131 configured to interchangeablylocate or move one or more of filter modules 131 into emission opticalpath 128. Optical system 100 may additionally comprise an excitationfilter assembly 132 comprising a plurality of filters, filtercomponents, elements, or modules 133, wherein excitation filter assembly132 is configured to interchangeably locate or move one or more offilter modules 133 into excitation optical path 126. Optical system 100may further comprise a first optical element 152 configured to directlight to optical sensor 118, a second optical element 154 configured todirect excitation light to, and/or emission light from, the biologicalsamples, a beamsplitter 160, and/or one or more optical windows 162.

In certain embodiments, sample processing system 200 comprises a carrieror support frame 202 configured to receive a sample holder 204. Sampleholder 204 comprises a plurality or array of cells 205 for containing acorresponding plurality or array of biological samples 115 that may beprocessed by sample processing system 200 and/or optical system 100.Cells 205 may be in the forms of sample wells, cavities, through-holes,or any other chamber type suitable containing and/or isolating theplurality of biological samples 115. For example, sample cells 205 maybe in the form of sample beads in a flow cell or discrete samplesdeposited on top of a substrate surface such as a glass or siliconsubstrate surface.

With additional reference to FIG. 4, sample holder 204 comprises 96sample cells 205 that are in the form of 96 sample wells 209 configuredto provide 96 isolated or distinct biological samples 115.Alternatively, sample holder 204 may comprise less than 96 well andsamples, for example, 48 wells and samples, or may contain more than 96wells, for example, 384 or more wells and samples. In certainembodiments, carrier 202 is configured to receive more than one sampleholder 204 for simultaneous processing by sample processing system 200and/or optical system 100.

Sample processing system 200 may further comprise a block or assembly210 for receiving sample holder 204 and a sample thermal or temperaturecontroller 211 for controlling and/or cycling the temperature ofbiological samples 115. In certain embodiments, sample holder 204includes all or a portion thermal controller 211. Sample processorsystem 200 may further comprise a thermally controlled or heated lid 212disposed about sample holder 204. Thermally controlled lid 212 may beconfigured to aid in controlling a thermal and/or humidity environmentof biological samples 115 or sample holder 204, for example, to aid inpreventing condensation from forming on samples 115 or optical elementsof sample holder 204. In certain embodiments, system 200 includes a setof different types or configurations of block 210 and/or different typesor configurations of thermally controlled lid 212, where each member ofthe set is configured for use with a different type or number of sampleholders 204 or carriers 202. Sample temperature controller 211 maycomprise all or a portion of heated lid 212 and/or hardware used tocontrol the temperature of, or heat flow into, heated lid 212.

Referring to FIG. 5, system 1000 may be additionally or alternativelyconfigured to receive and process a sample holder 304 comprising asubstrate 306 including a plurality of through-holes 309. In suchembodiments, through-holes 309 are configured to maintain isolated ordistinct biological samples 315 by capillary forces, for example, byforming through-holes to have an appropriately small diameter and/orthrough the use of hydrophilic and/or hydrophobic materials or coatings.Substrate 306 may further comprise an alphanumeric 320, a barcode 322,and/or similar symbol for identification or processing purposes.Referring to FIG. 6, sample holder 304 may further comprise an enclosureor case for protecting or sealing substrate 306 and the biologicalsamples contained in through-holes 309. The case may comprise a base 324and a cover 328 that are configured to seal substrate 306 between base324 and cover 328, for example, to reduce or prevent evaporation of thebiological samples. Cover 328 is made of a transmissive material andcomprises a top surface 330 and an opposing bottom surface 332 forproviding optical access to substrate 306. One or both surfaces 330, 332may comprise an antireflective coating, for example, to reduceretro-reflections of light from excitation beam 111 back toward opticalsensor 118. Additionally or alternatively, one or both surfaces 330, 332may be disposed at an angle relative to a front surface of substrate306, for example, to reduce retro-reflections of light from excitationbeam 111 back toward optical sensor 118. Referring to FIG. 7, one ormore sample holders 304 may be retained by or mounted on a carrier 302that is configured to be received by sample processing system 200. Inthe illustrated embodiment shown in FIG. 7, carrier 302 is configured toretain four or less sample holders 304. For clarity, not all thethrough-holes

In the illustrated embodiment shown in FIG. 5, each through-hole 309 hasa diameter of about 320 micrometers, a thickness of or about 300micrometer, and a volume of or about 33 nanoliters. Through-holes 309have a nominal spacing in the illustrated embodiment of about 500micrometers center to center. As discussed in greater detail below,optical system 100 may be configured to allow imaging and processing ofbiological samples contained in through-holes having in this range.Additionally or alternatively, system 1000 and/or optical system 100 isconfigured receive and process a sample holder 304 having smallerthrough-hole diameter and/or a smaller nominal spacing than in theillustrated embodiment of FIG. 5. For example, optical system 100 may beconfigured to allow system 1000 to receive and process a sample holder304 comprising through-holes having a diameter that is less than orequal to 250 micrometer and/or a volume that is less than or equal to 5nanoliters. Alternatively, optical system 100 may be configured to allowsystem 1000 to receive and process a sample holder 304 comprisingthrough-holes having a diameter that is less than or equal to 150micrometer and/or a volume that is less than or equal to one nanoliter.In certain embodiments, an initial sample or solution for a sampleholder, such as sample holders 204, 304, may be divided into hundreds,thousands, tens of thousands, hundreds of thousands, or even millions ofreaction sites, each having a volume of, for example, a few nanoliters,about one nanoliter, or less than one nanoliter (e.g., 10's or 100's ofpicoliters or less).

In the illustrated embodiments shown in FIGS. 4 and 5, sample holders204, 304 have a rectangular shape; however, other shapes may be used,such as a square or circular shape. In certain embodiments, a sampleholder such as sample holder 304 has a square shape and an overalldimension of 15 millimeter by 15 millimeter. In such embodiments, thesample holder may have an active area, region, or zone with a dimensionof 13 millimeter by 13 millimeter. As used herein, the terms “activearea”, “active region”, or “active zone” mean a surface area, region, orzone of a sample holder, such as the sample holders 204 or 304, overwhich reaction regions, through-holes, or solution volumes are containedor distributed. In certain embodiments, the active area of sample holder304 may be increased to 14 millimeter by 14 millimeter or larger, forexample, a 15 millimeter by 15 millimeter substrate dimension.

In the illustrated embodiment of FIG. 5, through-holes 309 may have acharacteristic diameter of or about 320 micrometer and a pitch of orabout 500 micrometers between adjacent through-holes. In otherembodiments, through-holes 309 have a characteristic diameter of orabout 75 micrometer and have a pitch of or about 125 micrometers betweenadjacent through-holes. In yet other embodiments, through-holes 309 havea characteristic diameter of that is less than or equal 75 micrometers,for example, a characteristic diameter that is less or equal to 60micrometers or less or equal to 50 micrometers. In other embodiments,through-holes 309 have a characteristic diameter that is less than orequal to 20 micrometers, less than or equal to 10 micrometers, or lessthan or equal to one micrometer. The pitch between through-holes may beless than or equal to 125 micrometers, for example, less than or equalto 100 micrometers, less than or equal to 30 micrometers, or less thanor equal to 10 micrometers.

In certain embodiments, sample holder 304 comprises a substrate having athickness between the opposing surfaces of sample holder 304 that is ator about 300 micrometer, wherein each through-hole 309 may have a volumeof 1.3 nanoliter, 33 nanoliters, or somewhere between 1.3 nanoliter and33 nanoliters. Alternatively, the volume of each through-holes 309 maybe less than or equal to one nanoliter, for example, by decreasing thediameter of through-holes 309 and/or the thickness of sample holder 304substrate. For example, each through-holes 309 may have a volume that isless than or equal to one nanoliter, less than or equal to 100picoliters, less than or equal to 30 picoliters, or less than or equalto 10 picoliters. In other embodiments, the volume some or all of thethrough-holes 309 is in a range from one nanoliter to 20 nanoliters.

In certain embodiments, the density of through-holes 309 is at least 100through-holes per square millimeter. Higher densities are alsoanticipated. For example, a density of through-holes 309 may be greaterthan or equal to 150 through-holes per square millimeter, greater thanor equal to 200 through-holes per square millimeter, greater than orequal to 500 through-holes per square millimeter, greater than or equalto 1,000 through-holes per square millimeter, or greater than or equalto 10,000 through-holes per square millimeter.

Advantageously, all the through-holes 309 with an active area may besimultaneously imaged and analyzed by an optical system. In certainembodiments, active area comprises over 12,000 through-holes 309. Inother embodiments, active area comprises at least 25,000, at least30,000, at least 100,000, or at least 1,000,000 through-holes.

In certain embodiments, through-holes 309 comprise a first plurality ofthe through-holes characterized by a first characteristic diameter,thickness, or volume and a second plurality of the through-holescharacterized by a second characteristic diameter, thickness, or volumethat is different than the first characteristic diameter, thickness, orvolume. Such variation in through-hole size or dimension may be used,for example, to simultaneously analyze two or more different nucleotidesequences that may have different concentrations. Additionally oralternatively, a variation in through-hole 104 size on a singlesubstrate 304 may be used to increase the dynamic range of a process orexperiment. For example, sample holder 304 may comprise two or moresubarrays of through-holes 309, where each group is characterized by adiameter or thickness that is different a diameter or thickness of thethrough-holes 309 of the other or remaining group(s). Each group may besized to provide a different dynamic range of number count of a targetpolynucleotide. The subarrays may be located on different parts ofsubstrate 304 or may be interspersed so that two or more subarraysextend over the entire active area of sample holder 304 or over a commonportion of active area of sample holder 304.

In certain embodiments, at least some of the through-holes 309 aretapered or chamfered over all or a portion of their walls. The use of achamfer and/or a tapered through-holes have been found to reduce theaverage distance or total area between adjacent through-holes 309,without exceeding optical limitations for minimum spacing betweensolution sites or test samples. This results in a reduction in theamount liquid solution that is left behind on a surface of substrate 304during a loading process. Thus, higher loading efficiency may beobtained, while still maintaining a larger effective spacing betweenadjacent solution sites or test samples for the optical system.

In certain embodiments, system 1000 is configured to receive and processdifferent types or numbers of block 210, carrier 202, and/or sampleholder 204. For example, Thus, system 1000 may be configured to receiveand process different sample holders 204 having different numbers ofwells 209. Thus, system 1000 may be configured to receive and processsample holders 204 containing 96 samples and sample holders 204containing 48 wells and/or 384 well or/or more than 384 wells.Additionally or alternatively, system 1000 may be configured to receiveand process different sample formats or container configurations. Forexamples, in addition to receiving a sample holder 204 comprising apredetermined number of wells, system 1000 may also be configured toreceive and process one or more sample holders 304 comprising theplurality of through-holes 309. In certain embodiments, system 1000 isconfigured to receive and process four different types of sampleholders. Some of the characteristics of wells or through-holes used inthese four sample holders are listed in Table 1 below.

TABLE 1 Characteristics of four sample holders according to anembodiment of the present invention. Sample Cell Number CharactersticCharacteristic Sample Holder Type of Cells Cell Volume Cell Diameter AWell  96 200 microliters 5 millimeters B Well 384  50 microliters 3millimeters C Cavity 384  2 microliters 3 millimeters D Through-hole 4 ×3072 0.033 microliters   0.35 millimeters

Referring again to FIGS. 1-3, optical sensor 118 may comprise one ormore photodetectors or photosensors, for example, one or morephotodiodes, photomultiplier tubes, or the like. Alternatively, opticalsensor 118 may comprise a one-dimensional or two-dimensionalphotodetector array 164, such as a charge-coupled device (CCD),complementary metal-oxide-semiconductor (CMOS), or the like. In theillustrated embodiment in FIG. 1, photodetector array 164 comprises atwo dimensional array of photosensitive pixels defining photosensitivesurface upon which an optical image or signal may be formed by emissionoptical system 125.

Excitation source 110 may be an excitation light source the produceselectromagnetic radiation that is primarily or exclusively within thevisible waveband of the electromagnetic spectrum. Excitation source 110may be a halogen lamp, a Xenon lamp, high-intensity discharge (HID)lamp, one or more light emitting diodes (LEDs), one or more laser, orthe like. In certain embodiments, excitation source 110 comprises aplurality of light sources having different emission wavelength rangesto excite different fluorescent dyes in biological samples 115, forexample, a plurality of LED light sources having different colors oremission wavelength ranges. In such embodiments, excitation filterassembly 132 may be omitted or may be incorporated for use with at leastsome of the different light sources to further limit the wavelengthrange of light or radiation reaching samples 115.

In certain embodiments, excitation source 110 comprises one or morebroadband or white light LED sources. For example, excitation source 110may comprise a high power, broadband source having at least 5 watts oftotal output optical power, at least 10 watts of output optical power,or at least 25 watts of output optical power. In such embodiments,excitation filter assembly 132 may be incorporated to limit or definethe spectral content of the radiation or light received by samples 115and/or sample holder 204, 304. The spectral content of the broadbandsource 110 may be configured to favorably provide more energy overwavelength ranges that, for example, correspond to probes or dyemolecules in samples 115 that are less efficient, are typically foundlower concentrations, or otherwise require more photonic energy thatother dyes contained in samples 115.

In a non-limiting example, in certain embodiments, excitation source 110comprises a single broadband LED having a total optical power of greaterthan 10 watts over the spectral range produced by the LED. The spectraloutput characteristics of such an excitation source are shown by thesolid line in the graphs shown in FIG. 8. The horizontal axiscorresponds to the wavelength of radiation emitted by the LED excitationsource 110, while the vertical axis is relative output intensity. The“relative intensity” for the plot in FIG. 8 is a percentage value thatis defined as 100 times the intensity measured at a given wavelengthdivided by the maximum intensity measured at any wavelength within rangeof wavelengths produced by the LED. For example, according to the plotin FIG. 8, the measured intensity out of the LED at a wavelength of 450nanometer is about 80 percent of the maximum intensity, where themaximum measured intensity occurs at an output wavelength of 457nanometers. By way of comparison, similar data for a halogen lamp usedin a prior art system is also shown in FIG. 8 as dashed lines. The setsof double lines with numeral in between indicate the approximatetransmission wavelength bands for the excitation filters shown in FIG.3.

For the illustrated embodiment shown in Table 1, the characteristic celldiameter and volume of sample holder D is much smaller than that ofsample holders A-C. As a result, a typical fluorescence signal producedby sample holder D is much smaller than a typical fluorescence signalproduced by sample holders A-C under similar conditions, for example,when using biological samples containing similar concentrations of abiological test sample and/or a fluorescent probe or reference dye. Forthese reasons, the halogen excitation source shown in FIG. 8 may notprovide sufficient intensity or power density for fluorescent probes ordyes excited by light in the wavelength range provided by filters 1-3 inFIG. 3.

In certain embodiments, fluorescent probes or dyes excited by light inthe wavelength ranges provided by excitation filters 1, 2, and 4 in FIG.3 are either more commonly used or are of greater importance than thoseexcited by light in the wavelength range provided by filters 3, 5,and/or 6, for example, in the wavelength range provided by eitherfilters 5 or 6. For example, in certain embodiments, the dyes FAM™(fluorescein amidite), VIC®, and ROX™ are used, which dyes arecommercially available from Life Technologies in Carlsbad, Calif. Insuch embodiments, excitation filter 1 is used to excite the dye FAM™,excitation filter 2 may be used to excite the dye VIC®, and excitationfilter 4 may be used to excite the dye ROX™. Additionally oralternatively, it may be that fluorescent probes or dyes excited bylight in the wavelength ranges provided by filters 3, 5, and/or 6 arenot used with all types of sample holders A-D and/or with all types ofsample holders 204, 304, for example, are not used with sample holders Dand/or sample holder 304. In such embodiments as these, an excitationsource 110 comprising an LED source having spectral characteristics thesame or similar to those shown in FIG. 8 has an unexpected beneficiallymeasurable or useful signal from all or most filters 1-6 for at leastsome sample holders 204, 304 (e.g., for all sample holders except sampleholder D in Table 1), even though (1) the spectral power or intensity ofthe LED source for fluorescent probes or dyes excited by light in thewavelength range provided by filters 5 and 6 is less than that for thehalogen source shown in FIG. 8, and (2) the spectral power or intensityof the LED source for fluorescent probes or dyes excited by light in thewavelength range provided by filters 5 and 6 is less than that forfluorescent probes or dyes excited by light in the wavelength rangeprovided by filters 1, 2, and/or 4. It has been discovered that, due tothe relatively large sample volumes provided by the sample cells insample holders A-C in Table 1, an LED source such as that characterizedin FIG. 8 is able to provide enough excitation energy to the biologicalsamples so that system 1000 is able to process the signals or imagesreceived by optical sensor 118.

Accordingly, it has been discovered that instrument or system 1000 canprocess biological samples to provide useful data using a broadband LEDthat produces light or radiation having a maximum intensity and/or powerdensity at a wavelength that is less than 600 nanometers and/or that isless than 550 nanometers. For example, instrument or system 1000 canprovide useful PCR data (e.g., qPCR and/or dPCR data) using such abroadband LED, such as that represented in FIG. 8. The result is aninstrument that can provide data, such as PCR data, over a wide range ofsample sizes and sample holder or cell formats, for example, all thesample sizes and sample cell formats listed in Table 1.

In certain embodiments, system 1000 includes an excitation source 110comprising an LED having a spectral profile characterized by a maximumintensity or output power at a wavelength that is less than a firstpredetermined wavelength or wavelength range, and an intensity or outputpower that is less than 50 percent the maximum value at a secondwavelength or wavelength range. For example, system 1000 may include anexcitation source 110 comprising an LED having a spectral profilecharacterized by a maximum intensity or output power at a wavelengththat is less than 550 or 600 nanometers and an intensity or output powerthat is less than 50 percent the maximum value at a wavelength of 650nanometer and/or 670 nanometers. In other embodiments, system 1000includes an excitation source 110 comprising an LED having a spectralprofile characterized by a maximum intensity or output power at awavelength that is less than 550 or 600 nanometers and an intensity oroutput power that is less than 30 percent or less than 20 percent themaximum value at a wavelength of 650 nanometer and/or 670 nanometers. Incertain embodiments, the system 1000 further comprise an emissionoptical system 125 that is able to provide useful biological data (e.g.,PCR data) for sample cells having a diameter of less than 500micrometer, less than 200 micrometers, or less than 100 micrometers thatcontain fluorescent probes or dye molecule that fluoresce at excitationwavelengths that are less than or equal to 560 nanometer, while alsobeing able to provide useful biological data (e.g., PCR data) for samplecells having a diameter of greater than 2 millimeters or greater than 3millimeters that contain fluorescent probes or dye molecule thatfluoresce at excitation wavelengths that are greater than or equal to620 nanometer or greater than or equal to 650 nanometers.

In certain embodiments, an instrument for biological analysis comprisesa sample processing system, an excitation source and a correspondingexcitation optical system, an optical sensor, and an emission opticalsystem. For example, the system may comprise all or portions of thesystem 1000 shown in FIG. 1, or a similar such system. The sampleprocessing system may be configured to retain a sample holder comprisingone or more sample cells. The excitation source may be configured toproduce one or more excitation beams that are directed by the excitationoptical system toward a plurality of samples retained by the sampleprocessing system. The instrument optical sensor may be configured toreceive emission beams from the one or more sample cells via theemission optical system. In such embodiments, the excitation source maybe characterized by a spectral function of optical output power orintensity of the excitation source verses wavelength of output power orintensity. The spectral function may be characterized by (a) a minimawavelength corresponding to a local minima value of the optical outputpower or intensity of the excitation source, (b) a first maximawavelength corresponding to a first local maxima of optical output poweror intensity of the excitation source, and (c) a second maximawavelength corresponding to a second local maxima of optical outputpower or intensity of the excitation source. The optical output power orintensity at the first local maxima is greater than the optical outputpower or intensity at any wavelength less than the minima wavelength,while the optical output power or intensity at the second local maximais greater than the optical output at any wavelength greater than theminima wavelength. The minima wavelength has a value that is between thefirst maxima wavelength and the second maxima wavelength. In certainembodiments, the optical output power or intensity is a relativeintensity of the excitation source, a relative power of the excitationsource, a relative luminous flux of the excitation source, or a radiantflux of the excitation source. For example, referring again to FIG. 8 asan example, a spectral function according to the above embodiment isillustrated in which (a) the minima wavelength is at or about 480nanometers and corresponds to a relative intensity of about 25 units,(b) the first maxima wavelength is at or about 460 nanometers andcorresponds to a relative intensity of about 100 units, and (c) thesecond maxima wavelength is at or about 550 nanometers and correspondsto a relative intensity of about 80 units.

When used in system 1000 according to embodiments of the presentinvention, another unexpected benefit of an LED excitation source 110 asdescribed in the previous paragraph and/or as illustrated in FIG. 8 isthat infrared (IR) emissions from excitation source 110 are much lowerthan, for example, a halogen light source like or similar to that shownin FIG. 8. Thus, embodiments of the present invention provide reduced IRnoise without the need extra optical element such as so-called “hotmirrors” to block IR emissions.

In certain embodiments, the output intensity, power, or energy ofexcitation source 110 may be varied depending on a condition or variablevalue, for example, depending on the type of sample holder used, size ofone or more reaction regions, experiment or run conditions of system orinstrument 1000, experiment or run conditions of optical system 100,experiment or run conditions of sample processing system 200, or thelike. For example, excitation source 110 may be an LED light source inwhich the output intensity, power, or energy is varied depending on oneor more of the conditions and/or variable values. In such embodiments,the output intensity, power, or energy of the LED may be varied byadjusting or changing a current or voltage driving the LED, and/or byadjusting or changing a duty cycle of the LED. In certain embodiments,the output intensity, power, or energy of excitation source 110 ischanged depending on the type of sample holder being used in system1000. For example, in certain embodiments, excitation source 110 may bean LED that is run at full output power, intensity, or energy—or at ahigher power setting output power, intensity, or energy—when sampleholder D from Table 1 is used. By contrast, the LED may be run at alower output power, intensity, or energy when a different sample holderis used, for example, sample holder A, B, or C, from Table 1 is used.Such an arrangement allows system 1000 to provide emission data for thesmaller sample volume sizes and/or lower sample concentrations thatoccur when sample holder A is used, while also avoiding a saturation ofoptical sensor 118 when other larger sample volumes and/or higher sampleconcentrations are used.

Referring again to FIG. 1, lens 152 is configured to form an image onphotodetector array 164, for example, by focusing collimated radiationentering from a particular direction to a spot or point, for example, toa diffraction limited spot or a nearly diffraction limited spot. Lens152 may be a simple lens, such as a plano-convex lens, plano-concavelens, bi-convex lens, bi-concave lens, meniscus lens, or the like.Alternatively, lens 152 may comprise a compound lens such as a doubletlens or triplet lens that may, for example, comprise different lensmaterials selected to correct or reduce a chromatic aberration. In otherembodiments, lens 152 comprises system of lenses such as a camera lenssystem or microscope objective, for example, a commercially availablecamera lens. The camera lens system may be a commercially availablecamera lens comprising a conventional lens system design, for example, adouble Gauss design, a Cooke triplet design, retrofocus lens design(e.g., Distagon lens design), a Tessar lens design, or the like.

Lens 154 may be a single field lens, for example, configured to providea telecentric optical system when combined with the remaining opticalelements of excitation optical system 120 and/or emission optical system125. In such embodiments, lens 154 may be a simple lens, such as aplano-convex lens, plano-concave lens, bi-convex lens, bi-concave lens,meniscus lens, or the like. Alternatively, lens 152 may comprise adoublet lens or triplet lens, for example, comprising different lensmaterial to correct for a chromatic aberration. Additionally oralternatively, lens 154 may comprise a Fresnel lens or a diffractiveoptical element, surface, or pattern. In certain embodiments, lens 154may comprise a lens system, for example, a field lens in combinationwith an additional lens or lenslet array configured to focus lightwithin a sample well of sample holder 204. The lenslet array maycomprise a Fresnel lens or a diffractive optical element, surface, orpattern. Examples of such lens configurations are also describe in U.S.Pat. No. 6,818,437, which is herein incorporated by reference in itsentirety as if fully set forth herein.

Referring to FIG. 9, in certain embodiments, heated lid 212 comprises alenslet array 166, for example, for use with sample holder 204 (e.g.,like sample holders A-C listed in Table 1) for focusing light fromexcitation beam 111 into a well or cavity of a sample holder, such assample holders 204 in the illustrated embodiment. With additionalreference to FIG. 10, heated lid 212 may additionally or alternativelycomprise an optical window 167 for providing thermal isolation orimproved thermal performance of the thermal environment in or around asample holder, such as sample holder 304 in the illustrated embodiment.In certain embodiments, convective current can be produced when window167 is not located as shown in FIG. 10. Such convective heat flow hasbeen found to result in higher temperature or thermal non-uniformity(TNU) in substrate 306, in sample holder 304, and/or between samples 315than may be acceptable in some applications. Accordingly, placement ofwindow 167 between lens 154 and sample holder 304 can decrease theamount of convective currents around sample holder 304 and lead to adecreases in TNU.

Optical window 167 may be used in addition to or in place of opticalwindow 162 shown in FIG. 1. Either or both windows 162, 167 may bedisposed parallel to a surface of sample holder 304 and/or perpendicularto optical axis 170. Alternatively, one or both windows 162, 167 may bedisposed at an angle relative to a surface of sample holder 304 and/orat an acute angle to optical axis 170, for example, to reduceretro-reflections of light from excitation beam 111 back toward opticalsensor 118. One or both windows 162, 167 may comprise an antireflectivecoating to reduce retro-reflections of light from excitation beam 111back toward optical sensor 118. The antireflective coating may be usedin addition to, or as an alternative to, tilting one or both windows162, 167. Thus, system 1000 is able to accommodate and provide usefulbiological data (e.g., PCR data) for sample holders having a diversityof optical requirements by providing heated lids 212 having differentoptical characteristics from one another and/or serving differingthermal requirements for sample holders, such as sample holders 204, 304(e.g., sample holders A-D listed in Table 1).

In certain embodiments, the combination of lenses or lens systems 152,154 is selected to provide a predetermined optical result or imagequality. For example, in order to reduce system cost or to simplify theemission optical system 125 design, lens 152 may comprise a commerciallyavailable camera lens. Such lenses can provide very high image quality(e.g., images with low chromatic and monochromatic aberration) undercertain viewing conditions. However, the careful balance of higher orderaberrations incorporated into such camera lens design used to providesuch high image quality can be disturbed with the introduction of otherlenses into an imaging system. For example, in the illustratedembodiment shown in FIG. 1, a field lens such as lens 154 is added toemission optical system 125. Lens 154 is common to both excitationoptical system 111 and emission optical system 125 to provide both agenerally more compact optical system and efficient transfer offluorescent energy from a sample to the detection system.

In prior art systems, a field lens having a plano-convex lens shape orfigure has been found to provide certain favorable characteristic inthis respect, for example, to provide a telecentric lens systemconfigured to provide even illumination over a large field of view.However, to provide an acceptably low level of optical aberrations, suchprior art systems also incorporate a custom camera lens design in orderto reduce overall system aberrations when used in combination theplano-convex field lens. In particular, due to the extended field ofview used to simultaneously image a large number of biological samples,the camera lens was designed to provide low amounts of field curvature.However, it has been discovered that the combination of a plano-convexlens with a conventional or commercially available camera lens canresult in large amounts of field curvature that are undesirable. It hasbeen further discovered that field curvature can be significantlyreduced by combining a biconvex field lens 154 with a conventional orcommercially available camera lens, as illustrated in FIG. 1. Thisresult is surprising, since a biconvex lens would normally be expectedto reduce overall image quality in a telecentric lens system. Forexample, it has been found that when lens 152 comprises a commerciallyavailable camera lens of a retrofocus design (e.g., a Distagon lensdesign), the amount of field curvature produced when field lens 154 is abiconvex lens is much smaller than the amount of field curvatureproduced when field lens 154 is a plano-convex lens.

Emission filter assembly 130 may comprise a first filter module 138characterized by a first optical power and a first filter 140 having afirst filter function or transmission range 140 a. In the illustratedembodiment, first filter function 140 a is shown as filter number 6 inthe table of FIG. 2 and is characterized by a first low-pass wavelength140L of 700 nanometers and a first high-pass wavelength 140H of 720 nm,so that light within this wavelength range is transmitted, or largelytransmitted, through the first filter 140, while light or otherelectromagnetic radiation outside this wavelength range is blocked, orsubstantially blocked, by first filter 140. The wavelengths listed inFIGS. 2 and 3 may represent the wavelengths at which the transmission ofa filter is one-half the maximum transmission of the filter over thetransmission wavelength range. In such cases, the difference betweenhigh-pass wavelength and the low-pass wavelength define a full width athalf maximum transmission (FWHM) range.

Emission filter assembly 130 also includes a second, and optionally athird, filter component, element, or module 142, 143. Second and thirdfilter modules 142, 143 are characterized by second and third filters145, 146 having a second and third filter functions or transmissionranges 145 a, 146 a. Either or both filter modules 142, 143 may have anoptical power that is the same as, or different from, the optical powerof first filter module 138. At least one of the filter modules 142, 143may have an optical power of zero, which power may in general be eitherpositive or negative. Filter functions 145 a, 146 a comprise second andthird low-pass wavelengths 145L, 146L second and third high-passwavelengths 145H, 146H, respectively, for example, as filter numbers 1and 5 in the table of FIG. 2. Second and third low-pass wavelengths145L, 146L may be different than the low-pass wavelength 140L and/or maybe different from one another. Similarly, second and third high-passwavelengths 145H, 146H may be different than the high-pass wavelength140H and/or may be different from one another. In the illustratedembodiment, the transmission wavelength bands for filters 140, 145, 146(wavelengths 140L to 140H, 145L to 145H, and 146L to 146H) do notoverlap; however, in other embodiments, there may be at least someoverlap in the wavelength bands between two or more of the filters inemission filter assembly 130. In certain embodiments, one or more offunctions 140 a, 145 a, 146 a may comprise a function that is differentthan the simple bandpass configuration illustrated in FIG. 2.

FIG. 3 illustrates various filters available for use with excitationfilter assembly 132. In FIG. 1, excitation filter assembly 132 comprisesthree filters, for example, filters 1, 2, and 6 in FIG. 3, which in usemay correspond to filters 1, 2, and 6 shown in FIG. 2 for emissionfilter assembly 130. At least some of the filter modules 133 ofexcitation filter assembly 132 may have non-zero optical powers, whichpower may in general be either positive or negative. Alternatively, allthe filter modules 133 may have zero or about zero optical power. Incertain embodiments, selection of a particular filter module 133 isassociated with a particular filter module 131 of filter assembly 130.Alternatively, filter modules 133, 131 may be selected independently ofone another.

In the illustrated embodiment shown in FIG. 1, only three filter modulesare shown for each filter assembly 130, 132; however, either or bothfilter assemblies may comprise more or less than three filter modules.For example, FIGS. 2 and 3 each show a total of 6 filters, each of whichfilters may be associated an optical power (not shown). In certainembodiment, either or both filter assemblies 130, 132 contain all sixfilters shown in the table shown in FIGS. 2 and 3, respectively.Alternatively, either or both filter assemblies 130, 132 may compriseless than six filters.

Filter functions 145 a, 146 a comprise respective second low-passwavelengths 145L, 146L that may be different than the first low-passwavelength 140L and may be different from one another. Each filter ofthe filters in emission filter assembly 130 or in excitation filterassembly 132 may comprise a transmission range of electromagneticradiation or light that is different and non-overlapping from theremaining filters of filter assembly 130 or filter assembly 132.Alternatively, two or more of the filters in filter assembly 130 or infilter assembly 132 may comprise transmission ranges of electromagneticradiation or light that at least partially overlap one another.

In certain embodiments, the optical power one more or more of filtermodules 131, or of each filter modules 133, is selected to compensatefor or reduce an optical aberration of the remaining optical elements ofemission optical system 125 or excitation optical system 120 over awavelength range of the filter being used. For example, in order toprovide a predetermined image resolution or quality for various of thefilter modules 131 at an image plane of optical sensor 118 or emissionoptical system 125, the optical powers of some or all of filter modules131 may be selected to compensate for or reduce a chromatic orsphero-chromatic aberration introduced by emission optical system 125over different filter wavelength ranges. Additionally or alternatively,one or more of filter modules 131 or of filter modules 133 may comprisea monochromatic aberration, such as spherical aberration, astigmatism,or coma, that is configured to alter, adjust, or reduce an overallaberration of emission optical system 125 or excitation optical system120.

In certain embodiments, the optical power or a monochromatic aberrationof one or more of filter modules 131 is configured to at least partiallycorrect or adjust an image or focus of sample holder 204 and/or of atleast some of the biological samples 115 in an image plane at or near adetection surface of optical sensor 118. For example, in the illustratedembodiment, the optical powers of filter modules 138, 142, 143 are alldifferent from one another, with third filter module 143 having anoptical power of zero or about zero. The optical power of filter modules138, 142 may be selected so that an effective focal length of emissionoptical system 125 is adjusted over the transmission wavelength range ofeach filter 138, 142 is the same or about the same as the effectivefocal length when filter 143 is located in the emission optical system125. Additionally or alternatively, the optical power of filter modules138, 142 may be selected so that the image quality produced whencorresponding filter 140, 145 is inserted into emission optical system125 is the same or similar to the image quality produced when filter 146is inserted into emission optical system 125. For example, the opticalpower for each filter module 131 may be selected so that images ofbiological samples 115 are the same size, or about the same size, foreach filter module 138, 142, 143. Additionally or alternatively, theoptical power for each filter module 131 may be selected so that amagnification and/or aberration of images of biological samples 115 arethe same, or about the same, for each filter module 131. In certainembodiments, two or more of the optical powers may be the equal to oneanother. In general filter modules 138 and/or 142 may have opticalpowers that are greater than zero or less than zero in order to providea desired correction or adjustment to the emission optical system 125and/or images produced therefrom.

Beamsplitter 160 may be configured to selectively reflect a large amountof emitted light or radiation from excitation source 110 that istransmitted through a selected excitation filter module 133 and thendirected toward sample holder 204, 304. For example, the coatedbeamsplitter 160 may comprise a dichroic reflector that is configured toreflect at least 95 percent or at least 99 percent of incident lighttransmitted through excitation filter module 133. The same coating forbeamsplitter 160 can additionally be configured to transmit a largeamount of emission light or radiation from biological samples 115, forexample, to transmit at least 90 percent or at least 95 percent of lightor radiation emitted by biological samples 115. In certain embodiments,a different beamsplitter 160 is associated with each different filtermodule 133, for example, by attaching the different beamsplitters 160 toexcitation filter assembly 132. In certain embodiments, only some of thebeamsplitters 160 are wavelength selective or dichroic beamsplitters,while others of beamsplitters 160 associated with some of excitationfilter modules 133 are not wavelength selective, for example, a 50/50beamsplitter that reflect 50 percent of incident radiation over a broadband of wavelengths. In such embodiments, excitation light or radiationnot reflected by a beamsplitter 160, but transmitted through thebeamsplitter 160, may be intercepted by an emission filter module 131and directed to optical sensor 118 in the form of noise.

In certain embodiments, optical system 100 comprises a plurality ofoptical modules, where each optical module comprises a beamsplitter 160and an excitation filter or filter module 133 and/or an emission filteror filter module 131. Each optical module may be inserted or removedfrom excitation optical path 126 and/or emission optical path 128. Insome embodiments, each module comprises a beamsplitter 160 that iscommonly mounted with one of the excitation filters or filter modules133. In such embodiments, a beamsplitter/excitation filter pair 160, 133may be inserted and removed from excitation optical path 126, whileemission filters or filter modules 131 may be inserted and removed fromemission optical beam path 128 independently of thebeamsplitter/excitation filter pairs 160, 133.

In certain embodiments, noise from excitation light or radiationtransmitted through a beamsplitter 160 is reduced by reducing the sizeof the corresponding emission filter module 131. However, the sizereduction of the corresponding emission filter module 133 may be limitedso as to avoid loss of signal from at least some of the biologicalsamples 115, 315, for example, due to vignetting effects on the moreperipherally located samples. It has been discovered that a reduction inexcitation radiation noise can be accomplished without significant lossof emission radiation signal by configuring the emission filters to havea shape that is the same as, or similar to, the shape of the area ofsample holder 204, 304 containing samples 115, 315. For example, it canbe seen in FIG. 4, 5, or 7 that a rectangular area is defined by anactive area over which one or more of sample holders 204, 304 containsamples or sample cells within the field of view of optical sensor 118.In such cases, it has been found that a rectangular shaped emissionfilter 140, 145, 146 or emission filter module 138, 142, 143 providesreduced noise from excitation radiation transmitted through beamsplitter160, without a significant loss of emission signal from samples 115, 315or uneven illumination from samples over the entire area of sampleholders 204, 304. In certain embodiments, the rectangular emissionfilter 140, 145, 146 or emission filter module 138, 142, 143 has thesame, or a similar, aspect ratio as that defined by an active area ofsample holders 204, 304, by carriers 202, 302, or by area of samples115, 315 that are within the field of view or field of regard of opticalsensor 118. For example, the aspect ratio of a rectangular emissionfilter (e.g., filter 140, 145, and/or 146) or filter module (e.g.,filter module 138, 142, or 143) may be selected to be within 1 percent,5 percent, 10 percent, or 20 percent of the aspect ratio of the activearea of a sample holder (e.g., sample holders 204 or 304) or of a groupof sample holders (e.g., the four sample holders 304 shown in FIG. 7).

During operation, biological samples 115 are disposed in a sampleholder, for example in sample holder 204, sample holder 304, or thelike. Biological samples 115 may include one or more nucleotidesequences, amino acid sequences, or other biological macromoleculesincluding, but not limited to, oligonucleotides, genes, DNA sequences,RNA sequences, polypeptides, proteins, enzymes, or the like. Inaddition, biological samples 115 may include other molecules forcontrolling or monitoring a biological reaction including, but notlimited to, primers, hybridization probes, reporter probes, quenchermolecules, molecular beacons, fluorescent dyes, chemical buffers,enzymes, detergents, or the like. Additionally or alternatively,biological samples 115 may include one or more genomes, cells, cellularnucleuses, or the like.

Once the biological samples are loaded, one or more sample holders areloaded or mounted within system 1000. In the illustrated embodimentshown in FIG. 1, one or more sample holders are mounted in to carrier202 or 302, which in turn is received by block 210 system 1000 and maybe subsequently covered or secured by heated lid 212. As discussed aboveherein, block 210 and heated lid 212 may be removably mounted or securedwithin system 1000, for example, so either or both may be exchanged foranother block or heated lid that is configured for use with a particularsample holder or carrier. Once the sample holder has been received bysample processing system 200, optical system 100 is used to monitor ormeasure one or more biological reactions or processes.

Emission optical system 125 of optical system 100 comprises an opticalaxis 170. A first emission beam 172 of emission beams 119 is emitted bya first biological sample located at or near optical axis 170. Firstemission beam 172 passes through emission optical system 125 such thatat least a portion of the electromagnetic radiation from the sampleproduces a first sample image 173 at or near photodetector array 164that is on or near optical axis 170. A second emission beam 174 ofemission beams 119 is simultaneously emitted by second biological samplelocated at or near an outer edge location of the array of biologicalsamples 115. Second emission beam 174 also passes through emissionoptical system 125 such that at least a portion of the electromagneticradiation from the sample produces a second sample image 175 at or nearoptical sensor 118 that is displace from optical axis 170. Emissionbeams 172, 174 may be fluorescence beams produced by different probemolecules contained in the two respective samples in response toexcitation beam 111. Depending upon the particular excitation filtermodule 133 selected, emission beams 172, 174 have a wavelength orwavelength range corresponding to the particular probe molecule that isexcited by radiation from excitation beam 111 that is transmitted by theselected excitation filter module 133. For example, when filter number 1in FIG. 3 may be used to filter radiation from excitation beam 111 andused in combination with emission filter number 1 in FIG. 2 (filter 146of filter module 143 in FIG. 1) to transmit radiation from emissionbeams 172, 174 onto photodetector array 164. As discussed above herein,the combination of lenses 152, 154 may be selected to form images fromemission beams 172, 174 that is low in monochromatic aberrations, and inparticular has a low amount of field curvature. A lateral distance(e.g., in a direction normal to optical axis 170) between the first andsecond samples may be compared to a lateral distance between thecorresponding images produced by emission optical system 125 todetermine a transverse magnification for the system when filter 146 isbeing used.

In certain embodiments, for radiation within the transmission range ofemission filter 146, first and second beams 172, 174 are collimated ornearly collimated when they leave lens 154 and form images at or nearphotodetector array 164 that have relatively low monochromaticaberrations and define a base system magnification. During use, emissionfilter assembly 130 may be subsequently moved (e.g., translated orrotated) so that emission filter module 143 and filter 146 are replacedby emission filter module 138 and filter 140 so the filter 140 (filternumber 6 in FIG. 2) now becomes part of the emission optical system 125,as illustrated in FIG. 1. Optionally, excitation filter number 1 in FIG.3 may also be replaced with excitation filter number 6 along excitationbeam path 111. As a result of chromatic aberrations, for radiationwithin the transmission range of emission filter 140, first and secondbeams 172, 174 are no collimated, but are divergent when they leave lens154. Thus, beams 172, 174 form images 173, 175 at or near photodetectorarray 164 that are further away from a principal plane of lens 152 thanthe images formed when filter 146 is present in emission optical system125. To correct or compensate for this effective change in focal lengthof emission optical system 125 over the transmission wavelength range offilter 140, a lens or optic 178 with a net positive optical power isincluded in filter module 138.

The added optical power to filter module 140 and emission optical system125 may be provided by a singlet lens 178, as shown in the illustratedembodiment of FIG. 1. The lens may be a plano-convex lens, plano-concavelens, bi-convex lens, bi-concave lens, meniscus lens, or the like.Alternatively, lens 178 may comprise a compound lens such as a doubletlens or triplet lens that may, for example, comprise different lensmaterials selected to correct or reduce a chromatic aberration. Optic178 may additionally or alternatively comprise a diffractive opticalelement. Optic 178 may be either a separate optical element, as shown inFIG. 1, or combined with filter 140 to form a single element. Forexample, optic 178 and filter 140 may be bonded together along a commonoptical face. Alternatively, optic 178 and filter 140 be formed togetherfrom a single substrate, for example, formed from a filter materialhaving one or both optical surfaces that are curved and/or contain adiffractive optical pattern. In certain embodiments, optic 178 islocated in a different part of emission optical system 125 than shown inFIG. 1, for example, on or proximal lens 154, window 162, orbeamsplitter 160, or at a location between beamsplitter 160 and emissionfilter assembly 130.

In addition to changing the effective focal length of emission opticalsystem 125, filter 140 may also result in a change in transversemagnification for the system. For example, even when lens 178 isincluded in filter module 138, the lateral distance between images 173,175 may be different when filter module 138 is used than when filtermodule 143 is used. In addition, the change from filter module 143 tofilter module 140 may introduce or alter various monochromaticaberration of emission optical system 125, for example, a sphericalaberration and/or field curvature. Accordingly, optic 178 or filtermodule 138 may be configured to at least partially correct or compensatefor such differences or changes in magnification and/or in one or moremonochromatic aberrations relative to when filter module 143 is used. Incertain embodiments, system 1000 or computer system 400 may includeimage processing instructions to at least partially correct orcompensate for changes in magnification and/or in one or moremonochromatic aberrations introduced by the use of filter module 138into emission optical system 125. The image processing instructions maybe used in combination with, or in place of, corrective optic 178 to atleast partially correct or compensate for changes in produces by the useof filter 140 in place of filter 146, including changes in effectivesystem focal length, magnification, chromatic aberrations, and/or one ormore monochromatic aberrations such as defocus, spherical aberrations,or field curvature.

In certain embodiments, each filter module 131 is disposed, in its turn,along the emission optical path 128 at a location where emission beam119, or some portion thereof, is either diverging or converging, wherebyone or more of filter modules 138, 142, 143 alters the amount ofdivergence or convergence to correct or adjust an effective focal lengthof emission optical system 125 and/or a spot size at an image plane ofemission optical system 125. In such embodiments, an optical power of atleast one of filter modules 138, 142, 143 is non-zero (i.e., eitherpositive or negative) over at least the transmission wavelength range orfilter function of corresponding filter 138, 142, 143.

In certain embodiments, the optical power of one or more of filtermodules 131, or one or more of filter modules 133, is greater than zeroand less than one Diopter. For example, the optical power of one or moreof filter modules 131, or one or more of filter modules 133, is greaterthan zero and less than or equal to one-third of one Diopter, less thanor equal to one-quarter of one Diopter, or less than or equal toone-eighth of one Diopter. Thus, optical power adjustment, while greaterthan zero, may be relatively small, so that only sight adjustments aremade in the optical characteristics of the emission optical system 125for at least some of the filters 140, 145, 146. Such slight adjustmentin optical power in the emission optical system 125 for differentfilters have been found to provide important optical corrections,resulting images created at optical sensor 118 that allow for bettercomparison between image data at different excitation and emissionconditions.

While most of the discussion above has related to emission opticalsystem 125 and the associated filter module 131, it will be appreciatedthat embodiments of the present invention also encompass similartreatment, where appropriate, of excitation optical system 120 and theassociated filter module 133.

In the illustrated embodiment shown in FIG. 1, a non-zero optical powerfor some of filter module 131, 133 is provided by a separate lens.Alternatively, a filter module 131, 133 may comprise a single opticalelement having both an optical power and filter transmission function.In certain embodiments, the single optic is made of a single material.Alternatively, two or more materials or elements may be adhered, joined,or bond together to form a filter module. In certain embodiments, theoptical power may be provided by a diffractive or holographic opticalelement or surface. The diffractive or holographic element or surfacemay be configured to reduce the size or thickness of a filter module.Additionally or alternatively, the diffractive or holographic element orsurface may be configured to introduce a chromatic aberration that isused to reduce a chromatic aberration produced by the remaining elementsof optical systems 125 or 120. In yet other embodiments, one or more offilter assemblies comprises a Fresnel lens or a curved mirror.

In certain embodiments, filter assembly 130 and/or 132 comprise acarrousel configuration in which different filter modules 131 or 133 arerotated into and out of the emission optical path 128 or excitationoptical path 126, respectively. In certain embodiments, filter assembly130 and/or 132 comprises interchangeable optical elements havingdiffering optical powers and interchangeable filters having differingfilter functions, wherein the optical elements and filters areindependently selectable from one another.

First optical element 152 is disposed near the optical sensor and isconfigured to provide images of samples 115 and/or sample holder 200.First optic element 152 may be a simple lens, such as a plano-convex orbi-convex lens, or a commercially available camera lens, such as aDouble Gauss lens, Distagon lens, Angenieux retrofocus lens, Cooketriplet, or the like. In the illustrated embodiment, filter modules 131are located between beamsplitter 160 and optical element 152, proximaloptical element 152. Second optical element 154 may be located nearsample holder 200 and be configured to provide a telecentric opticalsystem for illumination of the plurality of biological samples 115.

Referring to FIG. 11, computer system 400 may comprise variousdetection, data/image processing, and/or control operations or processesmay be implemented using hardware, software, firmware, or combinationsthereof, as appropriate. For example, some operations or processes maybe carried out using processors or other digital circuitry under thecontrol of software, firmware, or hard-wired logic. (The term “logic”herein refers to fixed hardware, programmable logic and/or anappropriate combination thereof, as would be recognized by one skilledin the art to carry out the recited functions.) Software and firmwarecan be stored on non-transitory computer-readable media. Additionally oralternatively, at least some operations or processes may be implementedusing analog circuitry, as is well known to one of ordinary skill in theart. Additionally, electronic memory or other storage, as well ascommunication components, may be employed in embodiments of the currentinvention.

Without limiting the scope of the current invention, the block diagramin FIG. 11 illustrates one embodiment of a computer system 400 forcarrying out processing functionality according to various embodimentsof system 1000. Computer system 400 may be utilized to control one ormore polymerase chain reaction (PCR), sequencing, and/or genotypinginstruments, or the like. Computing system 400 can include one or moreprocessors, such as a processor 404. Processor 404 can be implementedusing a general or special purpose processing engine such as, forexample, a microprocessor, controller or other control logic. Computingsystem 400 may include bus 402 or other communication medium ormechanism for communicating information, and processor 404 coupled withbus 402 for processing information.

Further, it should be appreciated that a computing system 400illustrated in FIG. 11 may be embodied in any of a number of forms, suchas a rack-mounted computer, mainframe, supercomputer, server, client, adesktop computer, a laptop computer, a tablet computer, hand-heldcomputing device (e.g., PDA, cell phone, smart phone, palmtop, etc.),cluster grid, netbook, embedded systems, or any other type of special orgeneral purpose computing device as may be desirable or appropriate fora given application or environment. Additionally, a computing system 400can include a conventional network system including a client/serverenvironment and one or more database servers, or integration withLIS/LIMS infrastructure. A number of conventional network systems,including a local area network (LAN) or a wide area network (WAN), andincluding wireless and/or wired components, are known in the art.Additionally, client/server environments, database servers, and networksare well documented in the art. According to various embodiments,computing system 400 may be configured to connect to one or more serversin a distributed network. Computing system 400 may receive informationor updates from the distributed network. Computing system 400 may alsotransmit information to be stored within the distributed network thatmay be accessed by other clients connected to the distributed network.

Computing system 400 also includes a memory 406, which can be a randomaccess memory (RAM) or other dynamic memory, coupled to bus 402 forstoring instructions to be executed by processor 404. Memory 406 alsomay be used for storing temporary variables or other intermediateinformation during execution of instructions to be executed by processor404. Computing system 400 further includes a read only memory (ROM) 408or other static storage device coupled to bus 402 for storing staticinformation and instructions for processor 404.

Computing system 400 may also include a storage device 410, such as amagnetic disk, optical disk, or solid state drive (SSD) is provided andcoupled to bus 402 for storing information and instructions. Storagedevice 410 may include a media drive and a removable storage interface.A media drive may include a drive or other mechanism to support fixed orremovable storage media, such as a hard disk drive, a floppy disk drive,a magnetic tape drive, an optical disk drive, a CD or DVD drive (R orRW), flash drive, or other removable or fixed media drive. As theseexamples illustrate, the storage media may include a computer-readablestorage medium having stored therein particular computer software,instructions, or data. In certain embodiments, storage device 410comprises one or more of memory 406 or ROM 408.

Additionally or alternatively, storage device 410 may include othersimilar instrumentalities for allowing computer programs or otherinstructions or data to be loaded into computing system 400. Suchinstrumentalities may include, for example, a removable storage unit andan interface, such as a program cartridge and cartridge interface, aremovable memory (for example, a flash memory or other removable memorymodule) and memory slot, and other removable storage units andinterfaces that allow software and data to be transferred from thestorage device 410 to computing system 400.

Computing system 400 can also include a communications interface 418.Communications interface 418 can be used to allow software and data tobe transferred between computing system 400 and external devices.Examples of communications interface 418 can include a modem, a networkinterface (such as an Ethernet or other NIC card), a communications port(such as for example, a USB port, a RS-232C serial port), a PCMCIA slotand card, Bluetooth, etc. Software and data transferred viacommunications interface 418 are in the form of signals which can beelectronic, electromagnetic, optical or other signals capable of beingreceived by communications interface 418. These signals may betransmitted and received by communications interface 418 via a channelsuch as a wireless medium, wire or cable, fiber optics, or othercommunications medium. Some examples of a channel include a phone line,a cellular phone link, an RF link, a network interface, a local or widearea network, and other communications channels.

Computing system 400 may be coupled via bus 402 to a display 412, suchas a cathode ray tube (CRT) or liquid crystal display (LCD), fordisplaying information to a computer user. An input device 414,including alphanumeric and other keys, is coupled to bus 402 forcommunicating information and command selections to processor 404, forexample. An input device may also be a display, such as an LCD display,configured with touchscreen input capabilities. Another type of userinput device is cursor control 416, such as a mouse, a trackball orcursor direction keys for communicating direction information andcommand selections to processor 404 and for controlling cursor movementon display 412. This input device typically has two degrees of freedomin two axes, a first axis (e.g., x) and a second axis (e.g., y), thatallows the device to specify positions in a plane. A computing system400 provides data processing and provides a level of confidence for suchdata. Consistent with certain implementations of embodiments of thepresent teachings, data processing and confidence values are provided bycomputing system 400 in response to processor 404 executing one or moresequences of one or more instructions contained in memory 406. Suchinstructions may be read into memory 406 from another computer-readablemedium, such as storage device 410. Execution of the sequences ofinstructions contained in memory 406 causes processor 404 to perform theprocess states described herein. Alternatively hard-wired circuitry maybe used in place of or in combination with software instructions toimplement embodiments of the present teachings. Thus implementations ofembodiments of the present teachings are not limited to any specificcombination of hardware circuitry and software.

The term “computer-readable medium” and “computer program product” asused herein generally refers to any media that is involved in providingone or more sequences or one or more instructions to processor 404 forexecution. Such instructions, generally referred to as “computer programcode” (which may be grouped in the form of computer programs or othergroupings), when executed, enable the computing system 400 to performfeatures or functions of embodiments of the present invention. These andother forms of non-transitory computer-readable media may take manyforms, including but not limited to, non-volatile media, volatile media,and transmission media. Non-volatile media includes, for example, solidstate, optical or magnetic disks, such as storage device 410. Volatilemedia includes dynamic memory, such as memory 406. Transmission mediaincludes coaxial cables, copper wire, and fiber optics, including thewires that comprise bus 402.

Common forms of computer-readable media include, for example, a floppydisk, a flexible disk, hard disk, magnetic tape, or any other magneticmedium, a CD-ROM, any other optical medium, punch cards, paper tape, anyother physical medium with patterns of holes, a RAM, PROM, and EPROM, aFLASH-EPROM, any other memory chip or cartridge, a carrier wave asdescribed hereinafter, or any other medium from which a computer canread.

Various forms of computer readable media may be involved in carrying oneor more sequences of one or more instructions to processor 404 forexecution. For example, the instructions may initially be carried onmagnetic disk of a remote computer. The remote computer can load theinstructions into its dynamic memory and send the instructions over atelephone line using a modem. A modem local to computing system 400 canreceive the data on the telephone line and use an infra-red transmitterto convert the data to an infra-red signal. An infra-red detectorcoupled to bus 402 can receive the data carried in the infra-red signaland place the data on bus 402. Bus 402 carries the data to memory 406,from which processor 404 retrieves and executes the instructions. Theinstructions received by memory 406 may optionally be stored on storagedevice 410 either before or after execution by processor 404.

It will be appreciated that, for clarity purposes, the above descriptionhas described embodiments of the invention with reference to differentfunctional units and processors. However, it will be apparent that anysuitable distribution of functionality between different functionalunits, processors or domains may be used without detracting from theinvention. For example, functionality illustrated to be performed byseparate processors or controllers may be performed by the sameprocessor or controller. Hence, references to specific functional unitsare only to be seen as references to suitable means for providing thedescribed functionality, rather than indicative of a strict logical orphysical structure or organization.

Computing system 400 may be accessible to an end user through userinterface 420, for example, via communications interface 418.Additionally, computer system 400 may provide data processing, displayand report preparation functions, for example, via display 412 and/orone or more input devices 414. All such instrument control functions maybe dedicated locally to the system 1000 and may provide remote controlof part or all of the control, analysis, and reporting functions.

Computer system 400 may additionally or alternatively, comprisefunctionality or capabilities for communicating with, and/orcontrolling, one or more processes, systems, or subsystems of system orinstrument 1000. For example, computer system 400 may comprise one ormore interfaces or communications with excitation source 110, excitationsource temperature controller 112, sample thermal controller 211,optical system 100, and/or one or more temperature sensors (e.g. an LEDtemperature sensor 422). The one or more interfaces or communicationswith optical system 100 may include, but is not limited to, opticaldetector 118 (e.g., for adjusting position, controlling gain, framerate, data collection rate, binning, or the like), filter assemblies130, 132 (e.g. for moving different filters into or out of an excitationor emission beam path), beamsplitter 160 (e.g. for moving differentbeamsplitter into or out of an excitation and/or emission beam path),optical element 152 (e.g., for adjusting an image focus and/or lensposition), or the like.

EXAMPLE

Without limiting the scope of the current invention, an instrument 1000according to in an exemplary embodiment was constructed that included anexcitation source 110 that comprised a broad-band LED that producedsignificant amounts of output power or intensity across a large portionof the visible spectrum. Instrument 1000 of the exemplary embodiment wasalso configured to receive and obtain image data from a variety ofsample holders 204, 304 that included sample holders A, B, C, and Dlisted in Table 1. Instrument 1000 of the exemplary embodiment alsocomprised a computer system 400 according to that shown in FIG. 11. Theoptical reader of the instrument 1000 of the exemplary embodiment wasgenerally arranged in accordance with the system shown in FIG. 1. Theoptical sensor 118 was a CCD array detector. A number of instruments1000 according to present exemplary embodiment were constructed andoperated to obtain data discussed below. Thus, reference to theexemplary embodiment of instrument 1000 may refer to an individualinstrument or the plurality of instruments used in providing resultinginstrument design.

The LED 110 of the exemplary embodiment was a nominally 50 W LEDproviding approximately 2000 Lumens of output over the visible waveband.The LED 110 had a relative intensity as shown in FIG. 8, comprising amaximum power output at a wavelength within the visible range of theelectromagnetic spectrum and having a power output that was at least 10percent of the maximum power output over a wavelength range from 450nanometers to 600 nanometers. Using filters with the characteristicslike those shown in FIGS. 1 and 2, instrument 1000 was configured toprovide the following “channels” for measuring fluorescence levels of atleast the dyes shown in Table 2.

TABLE 2 Dye channels for instrument 1000 in the exemplary embodiment.Nominal Nominal Example Excitation Emission Channel Dye(s) Wavelength(nm) Wavelength (nm) 1 FAM ™, SYBR ® 470 520 2 VIC ® 520 558 3 TAMRA ™,NED ™ 550 586 4 ROX ™ 580 623 5 LIZ 640 682 6 Joda-4 662 711

LED 110 produced sufficient output power to provide qPCR data forchannels 1, 2, and 4 of Table 2 when sample holder D was mounted intosystem 1000. However, it was found that under some conditions, CCDdetector 118 saturated when sample holders A, B, or C were mounted intosystem 1000, thus rendering at least some data unusable. To solve thisproblem, a calibration procedure for adjusting operation of the LED 110was developed. In certain embodiments, the calibration procedurecomprises:

-   -   Providing calibration target is placed inside the instrument.    -   Measuring an output from an optical sensor (e.g., 90th        percentile of image intensity in the exemplary embodiment) over        a first predetermined duty cycle of LED 110 (e.g., 20% and 40%        duty cycle in the exemplary embodiment), for example, using        optical sensor 118.    -   Performing an interpolation and/or extrapolation and calculating        a duty cycle to produce a predetermined nominal optical sensor        output (e.g., an ADU value of 2900 units in the exemplary        embodiment).    -   Adjusting the LED duty cycle to produce the calculated duty        cycle.    -   Measuring an output from an optical sensor.    -   If the measurement is within a predetermined tolerance (e.g.,        plus/minus 5% in the exemplary embodiment) of the predetermined        nominal optical sensor output, calibration is completed and        calibration data is stored.    -   If the measurement is not within the predetermined tolerance,        measuring an output from an optical sensor (e.g., 90th        percentile of image intensity in the exemplary embodiment) over        a second predetermined duty cycle of LED 110 (e.g., 30% and 50%        duty cycle in the exemplary embodiment).    -   Performing an interpolation and/or extrapolation and calculating        a duty cycle to produce the predetermined nominal optical sensor        output.    -   Adjusting the LED duty cycle to produce the newly calculated        duty cycle.    -   Measuring an output from the optical sensor.    -   If the measurement is within the predetermined tolerance of the        predetermined nominal optical sensor output, calibration is        completed and calibration data is stored.    -   If the measurement is not within the predetermined tolerance,        measuring an output from an optical sensor (e.g., 90th        percentile of image intensity in the exemplary embodiment) over        a second predetermined duty cycle of LED 110 (e.g., 40% and 60%        duty cycle in the exemplary embodiment).    -   Performing an interpolation and/or extrapolation and calculating        a duty cycle to produce the predetermined nominal optical sensor        output.    -   Adjusting the LED duty cycle to produce the newly calculated        duty cycle.    -   Measuring an output from the optical sensor.    -   If the measurement is within the predetermined tolerance of the        predetermined nominal optical sensor output, calibration is        completed and calibration data is stored.    -   If the measurement is not within the predetermined tolerance,        then report calibration failure.

The above calibration procedure was performed for sample holders A, B,and C of Table 1, providing a duty cycle and/or drive current of LED 110value for each. The calibration procedure accounted for randomvariations in power output and spectral characteristics existing betweendifferent individual LEDs 110 from the same manufacturer and sold underthe same model or part number. The LED calibration procedure provided animage signal at CCD detector 118 that was approximately the same acrossvarious instruments 1000 of the same design and construction, regardlessof LED brightness and spectral characteristics for the particular LED110 used in an individual instrument 1000.

Based on the calibration procedure, an LED a duty cycle or drive currentvalue was stored in memory 406 of instrument 1000 for each of the sampleholder types A, B, and C shown in Table 1. In general, it was found thatthe duty cycle or drive current value may be different for each of thesample holder types A, B, and C; however, in other embodiments, the dutycycle or drive current values may be the same for two or more sampleholder types. It was also found that a duty cycle of 100% could be usedfor sample holder D; however, in other embodiments, a duty cycle of lessthan 100% may be stored from sample holder D. In addition, it was foundthat the duty cycle or drive currents stored for each of sample holdersA, B, and C, and optionally D, for the instrument 1000 could be used inall similar instruments configured the same, or essentially the same, asthe instrument 1000 of the exemplary embodiment.

Alternatively, a calibration procedure may be performed on an individualinstrument 1000, so that the duty cycle or drive currents stored foreach of sample holders A, B, and C, and optionally D, are customized forthat particular instrument.

As a result of the spectrum characteristics of LED 110, it wasdiscovered that an average sample fluorescence measurement time could bereduced by conducting the calibration procedure based on calibrationdata collected using channel 2 from Table 2. For example, when thecalibration procedure was conducted based on calibration data collectedusing channel 1, it was discovered that more exposure time for otherfive filter channels was generally necessary during runs, leading tolonger running time. When the calibration procedure is based on channel2, only 2 different exposure time are necessary for data collected onchannels 2, 3, 4, 5 and 6, and 3 exposure times is necessary only forchannel 1. More than one exposure time is used to increase the dynamicrange of the data measurements made for a particular channel(excitation/emission wavelength band).

Regarding the use of more than one exposure time for channels 1-6, whenan end users runs, for example, a real time PCR, the sample volume canbe different from user to user, and run to run; similar for sampleconcentration. Therefore, it is desirable to design instrument 1000′ toprovide a range of conditions for sample volume and sampleconcentration. If a single exposure time is provided, then for highersample volumes and/or higher sample concentrations, detector 118 imagesmay be saturated. Conversely, for lower sample volumes and/or lowersample concentrations, detector 118 images may be too low relative tonoise levels. Thus, multiple exposure times may be used to extend thedynamic range of the system.

Regarding the use of channel 2 for calibration of LED 110, it ispossible to use any of the filter channels for this purpose. In theexemplary embodiment, the calibration target fluoresces with morestrongly at wavelength within the band for channels 1 and 2. Thus,channels 1 or 2 were preferred to perform calibration of LED 110. Indetermining which of channels 1 and 2 to use for LED 110 calibration,Table 3 shows the average quant intensity (from detector 118 images) forsample holder B for different LED's used in different instruments of thesame design and construction.

TABLE 3 Average Ch1 Ch2 Ch3 Ch4 Ch5 Ch6 Instr 16 1078444 906267 845201212568 918702 908710 21 1002206 509389 440369 123634 480315 470166 221020165 504365 366718 111163 538647 523789 28 1062476 360443 26989972708 333860 305083 29 1148692 304511 272999 70278 273376 287532 301217466 402402 299384 80078 367132 369524

If channel 1 were used for LED 110 calibration, then all instrumentswould have about same image signal for channel 1, but there a largevariation in image signal produced when using excitation filters foreach of channels 2 to 6. For example, referring to Table 4, usingchannel 1 for calibration, channel 2 would have a minimum Ch2/Ch1 ratioof 0.238 and a maximum Ch2/Ch1 ratio 0.84. Thus, the ratio of themaximum Ch2/Ch1 ratio to the minimum Ch2/Ch1 ratio in Table 4 is 3.17.The implication of this large variation is that three exposure timeswould be needed to provide the dynamic range covered using two exposuretimes for Ch1, based on calibration with Ch1. Similarly three exposuretimes would be needed for channel 3 to 6. Thus, a total of3.times.5+2=17 exposure times are needed when all six channel (i.e., allsix excitation/emission filter ranges) are used for measuring samplescontained in sample holder 204. The total time to provide emission datafor all six channels is directly related to the total number of exposuretimes.

TABLE 4 Instrument Ch1/Ch1 Ch2/Ch1 Ch3/Ch1 Ch4/Ch1 Ch5/Ch1 Ch6/Ch1 161.000 0.840 0.784 0.197 0.852 0.842 24 1.000 0.672 0.525 0.148 0.6450.692 19 1.000 0.531 0.486 0.136 0.506 0.507 21 1.000 0.506 0.439 0.1230.479 0.469 22 1.000 0.494 0.359 0.109 0.528 0.513 28 1.000 0.339 0.2540.068 0.314 0.287 30 1.000 0.331 0.246 0.066 0.302 0.304 29 1.000 0.2650.238 0.061 0.238 0.250 Ratio of 1.000 3.169 3.297 3.221 3.578 3.365max/min

If we use channel 2 to perform the LED 110 calibration, the result isshown in Table 5, based on the same set of instruments and LED's shownin Table 3.

TABLE 5 Instrument Ch1/Ch2 Ch2/Ch2 Ch3/Ch2 Ch4/Ch2 Ch5/Ch2 Ch6/Ch2 161.190 1.000 0.933 0.235 1.014 1.003 24 1 487 1.000 0.781 0.220 0.9591.028 19 1.883 1.000 0.916 0.256 0.953 0.955 21 1.967 1.000 0.865 0.2430.943 0.923 22 2.023 1.000 0.727 0.220 1.068 1.039 28 2.948 1.000 0.7490.202 0.926 0.846 30 3.025 1.000 0.744 0.199 0.912 0.918 29 3.772 1.0000.897 0.231 0.898 0.944 Ratio of 3.169 1.000 1.283 1.285 1.190 1.227max/min

Calibrating LED 110 based on channels 2-6 all have about the samevariation (bottom row of Table 5) for the various LED's tested. Onlychannel 1 has a relatively large variation for the various LED's tested(ratio of max over min is 3.772/1.19=3.17). Thus, 3 exposure times areneeded for channel 1 to provide the same dynamic range as provided usingtwo exposure times for channels 2-6. As a result, a total of 2×5+3=13exposure times are needed, as compared to 17 exposure times when channel2 is used to calibrate LED 110. Thus, it has been discovered that thetotal time to provide emission data for all six channel may be reducedby using channel 2 to calibrate LED 110, instead of channel 1. Referringto the spectral function of LED 110 that is shown in FIG. 8, it has beenrealized that the reason why channel 2 to 6 change with about same ratefrom LED to LED is that they fall in the broad spectrum peak. Bycontrast, channel 1 falls in a narrow peak, and shows different changerate from other channels from LED to LED. It has been discovered thatthis is a reason why calibration of LED 110 using channel 2 results in alower number of image integration times to provide about the samedynamic range for six of channels 1-6.

It was discovered that the thermal performance, power output, andspectral characteristics of LED 110 may vary in the instrument 1000,depending on which of sample holders A, B, C, or D was used. A variationin these parameters of between different instruments was also found dueto random variations in LED characteristics between different individualLEDs 110 from the same manufacturer and sold under the same model orpart number. In order to reduce the LED performance variation betweensample holder types in a single instrument 1000, between differentinstruments 1000 of the same design and construction, and/or betweendifferent LEDs in the same instrument, an LED thermal calibrationprocedure was developed.

Referring to FIGS. 12-14, various instruments 1000 according to theexemplary embodiment were run under different conditions to determine anominal target temperature, a low temperature limit, and a hightemperature limit. Excitation source temperature control 112 comprised afan that was configured to maintain LED 110 at a constant temperature orwithin a predetermined temperature range. Instrument 1000 was configuredto operate over a range of environmental conditions in which the ambienttemperature in which the instrument was operated may be between 15degrees Celsius and 30 degrees Celsius. It will be appreciated thatembodiments of the present invention may be configured to operate withinother temperature ranges, for example, between 10 degrees and 30 degreesCelsius, between 15 degrees and 35 degrees Celsius, between 20 degreesand 25 degrees Celsius, or the like. It will also be appreciated that inother embodiments, excitation source temperature control 112 maycomprise other sources of controlling the LED temperature, for example,the use of a Peltier device or a liquid temperature controller.

In the current embodiment, instrument 1000 was run by operating the LEDusing different conditions, including different sample holder types(sample holders A, B, C, or D from Table 1), different instrumentenvironment temperature (15 degrees Celsius and 30 degrees Celsius), anddifferent fan conditions (fan off (Fan DC=0) or 90 percent maximum fandrive voltage (Fan DC=900). The performance of different instruments1000 having different LEDs 110 is shown in FIGS. 12-14. FIG. 12represents data obtained using sample holder A. FIG. 13 represents dataobtained using sample holders B or C. FIG. 14 represents data obtainedusing sample holder D. Data points on the right side of each plotrepresent data obtain with instrument 1000 in a 15 degree Celsiusenvironment, while data points on the left side of each plot representdata obtain with instrument 1000 in a 30 degree Celsius environment. Thedata points on the right side of the connecting lines is for the fan offcondition, while data points on the left side of the connecting lines isfor a fan drive voltage of 90 percent of the maximum.

Based on the data shown in FIGS. 12-14, suitable values for a nominaltarget temperature, a low temperature limit, and a high temperaturelimit for the different sample holders A, B, C, D are shown in Table 6.These values were used to control the temperature of the LED for thevarious sample holders 204 used in a way that reduced variations insystem performance and maintained a more consistent performance over adesired range of environmental operation conditions.

TABLE 6 Relevant temperature values for the exemplary embodiment ofinstrument 1000. Nominal Low High Sample Target Temperature TemperatureHolder Temperature Limit Limit A 30.5° C. 23° C. 38° C. B/C   36° C. 30°C. 42° C. D   50° C. 35° C. 65° C.LED Temperature Control

Based on the Example above, in certain embodiments, system or instrument1000 is configured so that sample processing system 200 can receive,retain, or hold a first sample holder 204 (e.g., sample holders A, B, C,or D from Table 1) comprising a first plurality of sample cells or unitsconfigured to hold a biological sample. Sample processing system 200 isalso configured to receive, retain, or hold a second sample holder 204(e.g., a different one of sample holders A, B, C, or D from Table 1)comprising a second plurality of sample cells or units. For clarity, thecurrent embodiment of system or instrument 1000 will be referred to assystem or instrument 1000′, where it will be appreciated that theelements, features, and/or embodiments discussed above in relation tosystem or instrument 1000, where appropriate, may be incorporated intosystem or instrument 1000′, or vice versa. System 1000′ is configured toretain only one sample holder at a time or to retain a group of sampleholders at a time that are all of the same type and construction. Thefirst and second sample holders 204 are different from one another in atleast one physical aspect. For example, a number, size, dimension, orvolume of the sample cells for the first sample holder 204 may bedifferent than that of the sample cells for sample holder 204.Additionally or alternatively, the form or structure of sample cells forthe first sample holder 204 may be different from that of sample cellsfor the second sample holder 204 (e.g., each may be made of a differentmaterial, or one of the sample holders may comprise through-holes thathold a liquid sample via capillary forces, while the other sample holdermay comprise a microtiter plate comprising a plurality of wells or amicrofluidics card comprising a plurality of sample chambers that areloaded via network of liquid flow channels). Sample processing system200 may be configured to also retain one or more additional sampleholders 204 (or sets of the same sample holder 204) each comprising aplurality of sample cells, wherein the number of the sample cells in theadditional sample holder or a characteristic dimension of the samplecells in the additional sample holder is different as compared to eitherthe first sample holder 204 or the second sample holder 204.

System 1000′ further comprises excitation source 110 and excitationsource temperature controller 112 including an excitation temperaturesensor 422, where excitation temperature sensor 422 is thermally coupledto the excitation source 110 so as to allow a temperature of theexcitation source 110 to be measured or determined. System 1000′ alsoincludes an electronic processor 404 and a memory 406 and/or storagedevice 410 that includes data comprising a first target temperature forfirst sample holder 204 and a second target temperature for the secondsample holder 204 that is different from or unequal to the value of thefirst target temperature. A memory or storage device 406, 410 may alsocomprise instructions for execution by processor 404 to control a systemtemperature to the first target temperature when the first sample holderis retained by the instrument and to control a system temperature to thesecond target temperature when the first sample holder is retained bythe instrument.

Excitation source 110 may be an LED, for example, as disclosed above inthe exemplary embodiment.

A memory or storage device 406, 410 may also comprise instructions forexecution by processor 404 to determine if a target temperature (e.g.,the first or second target temperature discussed above) of theexcitation source can be maintained for a retained sample holder 204(e.g., the first or second sample holders 204 discussed above).Referring to FIG. 15 a method 500 according an embodiment of the presentinvention includes a module 505 comprising selecting or determining atarget temperature. Method 500 also includes a module 510 comprisingoperating excitation source 110 for a predetermined period of time, T.Method 500 also includes a module 515 comprising reading one or moretemperatures from temperature sensor 422 during time T. Method 500 alsoincludes a module 520 comprising determining if one or more of thesensed temperatures meet predetermined criteria. Method 500 alsoincludes a module 525 comprising taking a first action if thepredetermined criteria are not meet and a module 530 taking a secondaction if the predetermined criteria are meet. FIG. 16 is an exemplaryflow chart 500′ of a specific embodiment of method 500.

The above presents a description of the best mode contemplated ofcarrying out the present invention, and of the manner and process ofmaking and using it, in such full, clear, concise, and exact terms as toenable any person skilled in the art to which it pertains to make anduse this invention. This invention is, however, susceptible tomodifications and alternate constructions from that discussed abovewhich are fully equivalent. Consequently, it is not the intention tolimit this invention to the particular embodiments disclosed. On thecontrary, the intention is to cover modifications and alternateconstructions coming within the spirit and scope of the invention asgenerally expressed by the following claims, which particularly pointout and distinctly claim the subject matter of the invention.

The following list of co-pending U.S. applications are hereinincorporated by reference in their entirely as if fully set forthherein:

-   -   International Patent Application No. PCT/US2012/058107, filed        Sep. 28, 2012.    -   U.S. Provisional Patent Application No. 61/541,453, filed on        Sep. 30, 2011.    -   U.S. Provisional Patent Application No. 61/541,515, filed on        Sep. 30, 2011.    -   U.S. Provisional Patent Application No. 61/541,342, filed on        Sep. 30, 2011.    -   U.S. Design patent application Ser. No. 29/403,049, filed on        Sep. 30, 2011.    -   U.S. Design patent application Ser. No. 29/403,059, filed on        Sep. 30, 2011.    -   U.S. Provisional Patent Application No. 61/541,495, filed on        Sep. 30, 2011.    -   U.S. Provisional Patent Application No. 61/541,366, filed on        Sep. 30, 2011.    -   U.S. Provisional Patent Application No. 61/541,371, filed on        Sep. 30, 2011.    -   U.S. Provisional Patent Application No. 61/564,027, filed on        Nov. 28, 2011.    -   U.S. Provisional Patent Application No. 61/660,343, filed Jun.        15, 2012.    -   U.S. Provisional Patent Application No. 61/768,367, filed Feb.        22, 2013.

What is claimed is:
 1. An instrument for biological analysis,comprising: a sample processing system configured to interchangeablyreceive, at a sample processing region configured to be thermallycontrolled, sample holders of differing sample holder formats, thesample processing system configured to receive one or more sampleholders having a given sample holder format at a time, wherein thediffering sample holder formats differ in one or both of (a) a number ofsample cells configured to retain individual volumes of sample and (b) acharacteristic dimension of the sample cells; an excitation sourceoperably coupled to the sample processing system and configured toproduce electromagnetic radiation configured to excite sample retainedin one or more sample holders receivable at the sample processingregion; an excitation source temperature controller in thermalcommunication with the excitation source and configured to regulate anoperating temperature of the excitation source; and an electronicprocessor configured to: receive input of differing operatingtemperature ranges respectively associated with the differing sampleholder formats, and execute instructions to control the excitationsource temperature controller to regulate the operating temperature ofthe excitation source to be within a given operating temperature rangeof the differing operating temperature ranges based on the given sampleholder format of one or more sample holders receivable at the sampleprocessing region of the sample processing system.
 2. The instrument ofclaim 1, further comprising a temperature sensor configured to senseinformation about the operating temperature of the excitation source,wherein the electronic processor is configured to receive theinformation about the operating temperature from the temperature sensor.3. The instrument of claim 2, wherein the electronic processor isfurther configured to execute instructions to: operate the excitationsource to produce electromagnetic radiation having a predetermined poweror intensity; receive the information about the operating temperaturefrom the temperature sensor a plurality of times over a predeterminedtime period; and determine if the operating temperature of theexcitation source can be maintained within the given operatingtemperature range.
 4. The instrument of claim 3, wherein the electronicprocessor is further configured to execute instructions to set a flag,produce an alarm, send a warning signal, or halt an operation of theinstrument, or any combination thereof, in response to a determinationthat the operating temperature of the excitation source cannot bemaintained within the given operating temperature range.
 5. Theinstrument of claim 1, wherein the excitation source is a light emittingdiode.
 6. The instrument of claim 1, wherein the excitation source is alight emitting diode comprising a maximum power output at a wavelengthwithin the visible range of the electromagnetic spectrum, the lightemitting diode having a power output that is at least 10 percent of themaximum power output over a wavelength range from 450 nanometers to 600nanometers.
 7. The instrument of claim 1, wherein the differingoperating temperature ranges comprise a range from a low temperaturelimit representative of a minimum operating temperature of theexcitation source and a high temperature limit representative of amaximum operating temperature of the excitation source, and a nominaltemperature that is between the low temperature limit and the hightemperature limit.
 8. The instrument of claim 1, wherein the differingoperating temperature ranges are predetermined based on a performance ofa plurality of different instruments, or of a plurality of differentexcitation sources in one or more instruments, or both.
 9. Theinstrument of claim 1, wherein the excitation source comprises aplurality of light emitting diodes (LEDs), the plurality of LEDscomprising a first LED configured to emit the electromagnetic radiationat a wavelength that excites a first dye and a second LED configured toemit the electromagnetic radiation at a wavelength that excites a seconddye.
 10. The instrument of claim 9, wherein the first dye comprises afirst fluorescent dye and the second dye comprises a second fluorescentdye.
 11. The instrument of claim 1, wherein the electromagneticradiation has an emission spectrum comprising one or more wavelengths inthe visible waveband of the electromagnetic spectrum.
 12. The instrumentof claim 1, further comprising one or more optical components havingoptical power, wherein the one or more optical components are arrangedin a path of the electromagnetic radiation produced by the excitationsource and to direct the electromagnetic radiation to the sampleprocessing region of the sample processing system.
 13. The instrument ofclaim 12, wherein the one or more optical components comprises a filterarranged to filter and transmit the electromagnetic radiation producedby the excitation source.
 14. The instrument of claim 13, wherein thefilter is configured to transmit the electromagnetic radiation at awavelength sufficient to excite a dye.
 15. The instrument of claim 14,wherein the dye comprises a fluorescent dye.
 16. The instrument of claim1, further comprising one or more optical sensors arranged to receiveelectromagnetic radiation emitted from the sample processing region ofthe sample processing system.
 17. The instrument of claim 1, wherein thesample processing system further comprises: a sample block at the sampleprocessing region, and a thermal controller in thermal communicationwith the sample block, the thermal controller configured to alter atemperature of the sample block.
 18. The instrument of claim 1, furthercomprising one or more data storage devices in communication with theelectronic processor, wherein the one or more data storage devices storedata comprising the differing operating temperature ranges and theinstructions the electronic processor is configured to execute, andwherein the electronic processor is configured to receive the input fromthe one or more data storage devices.
 19. The instrument of claim 1,wherein the differing sample holder formats comprise sample cells chosenfrom wells and through-holes, respectively.